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目的构建尿激酶型纤溶酶原激活因子(uPA)原核表达质粒,并在大肠埃希菌BL21中表达,为进一步研究uPA奠定基础。方法应用逆转录RT-PCR,从人肝细胞cDNA中扩增人尿激酶型纤溶酶原激活因子基因序列,与原核表达质粒pET32a重组,获得表达质粒uPA-pET32a。用氨苄青霉素平板筛选转化子,双酶切与DNA测序进行鉴定,用IPTG诱导表达,并用Western blotting进行鉴定。结果从肝细胞cDNA中扩增的uPA基因片段长1296bp,酶切及DNA测序证实uPA-pET32a重组质粒构建正确,表达融合蛋白分子量约为68900Mr,经Western blotting证实为目的蛋白。结论成功构建了重组原核表达质粒uPA-pET32a,并能在大肠埃希菌内表达,所表达的融合蛋白分子量大小与预期的相一致,为进一步的研究奠定了基础。
Objective To construct a prokaryotic expression plasmid of urokinase-type plasminogen activator (uPA) and express it in Escherichia coli BL21, which lays the foundation for further study of uPA. Methods The human urokinase-type plasminogen activator gene sequence was amplified from human hepatocyte cDNA by reverse transcription-polymerase chain reaction (RT-PCR) and cloned into the prokaryotic expression plasmid pET32a to obtain the expression plasmid uPA-pET32a. Transformants were screened by ampicillin plate, double digested with DNA sequencing and identified by IPTG, and identified by Western blotting. Results The fragment of uPA gene amplified from hepatocyte cDNA was 1296bp in length. Enzyme digestion and DNA sequencing confirmed that uPA-pET32a recombinant plasmid was constructed correctly. The molecular weight of the fusion protein was about 68,900Mr, which was confirmed by Western blotting as the target protein. Conclusion The recombinant prokaryotic expression plasmid uPA-pET32a was successfully constructed and expressed in Escherichia coli. The molecular weight of the expressed fusion protein was in accordance with the expectation, which laid the foundation for further study.